Abnormal and exaggerated deposition of extracellular matrix is the hallmark of all fibrotic diseases, including liver, pulmonary, kidney or cardiac fibrosis. The spectrum of affected organs, the progressive nature of the fibrotic process, the large number of affected persons, and the absence of effective treatment pose an enormous challenge when treating fibrotic diseases.
In an attempt to propose new therapeutic strategies for the treatment of fibrotic diseases, the inventors found that 2-[(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenyl]ethanoate (Nitazoxanide—NTZ), a synthetic antiprotozoal agent or its deuterated derivatives or its active metabolite 2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide (Tizoxanide, known as TZ) in combination with statins show synergistic antifibrotic activities. Moreover, the evaluation of NTZ combined to a statin in a liver injury model revealed its capacity to reduce circulating bile acid concentration, thus reflecting the synergistic potential of this combination to treat both cholestatic (such as PBC and PSC) and fibrotic diseases.
NTZ, first described in 1975 (Rossignol and Cavier, 1975), was shown to be highly effective against anaerobic protozoa, helminths, and a wide spectrum of microbes including both anaerobic and aerobic bacteria (Rossignol and Maisonneuve, 1984 Dubreuil, Houcke et al., 1996; Megraudd, Occhialini et al., 1998; Fox and Saravolatz, 2005; Pankuch and Appelbaum, 2006; Finegold, Molitoris et al., 2009). It was first studied in humans for the treatment of intestinal cestodes (Rossignol and Maisonneuve, 1984) and it is now licensed in the United States (Annie®, Romark laboratories) for the treatment of diarrhea caused by the protozoan parasites Crystosporidium parvum and Giardia intestinalis. NTZ has also been widely commercialized in Latin America and in India where it is indicated for treating a broad spectrum of intestinal parasitic infections (Hemphill, Mueller et al., 2006). The proposed mechanism of action by which NTZ exerts its antiparasitic activity is through the inhibition of pyruvate:ferredoxin oxidoreductase (PFOR) enzyme-dependent electron transfer reactions that are essential for anaerobic metabolism (Hoffman, Sisson et al., 2007). NTZ also exhibited activity against Mycobacterium tuberculosis, which does not possess a homolog of PFOR, thus suggesting an alternative mechanism of action. Indeed, the authors showed that NTZ can also act as an uncoupler disrupting membrane potential and intra-organism pH nomeostasis. (de Carvalho, Darby et al., 2011).
The pharmacological effects of NTZ are not restricted to its antiparasitic or antibacterial activities and in recent years, several studies revealed that NTZ can also confer antiviral activity (Di Santo and Ehrisman, 2014; Rossignol, 2014). NTZ interferes with the viral replication by diverse ways including a blockade in the maturation of hernagglutinin (influenza) or VP7 (rotavirus) proteins, or the activation of the protein PKR involved in the innate immune response (for a review, see (Rossignol, 2014)). NTZ was also shown to have broad anticancer properties by interfering with crucial metabolic and prodeath signaling pathways (Di Santo and Ehrisman, 2014).
Statins (3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) are commonly prescribed as medications for the treatment of hypercholesterolemia and for the prevention of cardiovascular disease.
There are currently 7 unique prescribed statins that include pravastatin, simvastatin, and lovastatin (which are naturally-derived from fungal fermentation) and a second group of chemically synthesized statins composed of fluvastatin, atorvastatin, rosuvasatin and pitavastatin. Although all statins contain a dihydroxy-heptanoic acid HMG-CoA like moiety, which competes for binding to HMG-CoA reductase, each statin is unique and exhibits significant differences in chemical structure, potency (ex. IC50 for HMG-CoA reductase inhibition), tissue penetration and retention, half-fife, metabolism and elimination, drug-drug interactions, and safety. The mechanisms involved in the beneficial effects of statins on the prevention of cardiovascular disease have been largely attributed to the ability of these agents to inhibit cholesterol biosynthesis. Owing to the fact that 60-70% of serum cholesterol is derived from hepatic biosynthesis and that HMG-CoA reductase is the crucial, rate-limiting enzyme in the cholesterol biosynthetic pathway, it is not surprising that inhibition of this enzyme results in a dramatic reduction in circulating LDL-Cholesterol. Moreover, reduction of LDL-cholesterol leads to upregulation of hepatic LDL receptors and increase of LDL clearance. Both clinical and experimental data suggest that the sum of benefits from statin therapy may extend well beyond their favorable effects on serum cholesterol levels. These cholesterol-independent effects, described as pleiotropic effects of statins, are related to the reduced formation of isoprenoids. Indeed, inhibition of HMG-CoA reductase results not only in deprivation of intracellular mevalonate but also several downstream isoprenoid derivatives including farnesyl pyrophosphate (FPP) and geranylgeranylpyrophosphate(GGPP). Both FPP and GGPP are required for posttranslational prenylation of a number of proteins (approximately 2% of total cellular proteins, (Wang, Liu et al., 2008)). Protein isoprenylation enables proper subcellular localization and trafficking of intracellular molecules. For example, non-isoprenylated GTPases remain cytosolic whereas isoprenylated GTPases harbour a FPP or GGPP lipid attachment that permits insertion and anchorage into the cell membrane, and subsequently participate in signal transduction. Therefore, inhibiting isoprenylation results in the inactivation of the small GTPases (ex Rho, Ras, Rac and Cdc42) which are essential in many cellular events (intracellular signal transduction, cellular proliferation, inflammation, motility, (for a review see (McFarlane, Muniyappa et al., 2002; Zhou and Liao, 2009; Yeganeh, Wiecher et al., 2014; Kavalipati, Shah et al., 2015)). Since it has been demonstrated that the Rho GTPase and its target protein Rock are involved in the activation/differentiation of fibroblasts into myofibroblasts (Ji, Tang et al., 2014), a key event in the fibrotic process, several studies were conducted with statins to evaluate their antifibrotic properties in different pathological models. Simvastatin was shown to reduce the expression of fibrotic markers in both human and rat HSC and to confer antifibrotic properties in various animal models of fibrosis (Rombouts, Kisanga et al., 2003; Watts, Sampson et al., 2005; Wang, Zhao et al., 2013, Marrone, Maeso-Diaz et al., 2015). As well, Pitavastatin (Miyaki, Nojiri et al., 2011) and Fluvastatin (Chong, Hsu et al., 2015) were able to reduce fibrosis in the CDAA diet-induced NAFLD/NASH model. These beneficial effects of statins are not restricted to liver fibrosis. Indeed, it was demonstrated that Atorvastatin was significantly potent against bleomycin-induced lung fibrosis (Zhu, Ma et at, 2013) while Simvastatin was shown to inhibit expression of fibrotic markers in fibroblasts derived from human fibrotic lung. (Watts, Sampson et al., 2005).
In this invention, using a phenotypic screening assay to identify potential antifibrotic agents, it was discovered that NTZ or its deuterated derivatives or its active metabolite TZ, in combination with a statin, interferes, in an additive or synergistic manner, with the activation of myofibroblasts. This effect was totally unexpected in view of the properties previously reported for these molecules. Combination of NTZ or its deuterated derivatives or TZ with a specific statin appears as a potent therapeutic strategy for diverse types of fibrotic diseases. Moreover, the evaluation of NTZ or derivatives thereof in combination with a specific statin revealed an unexpected synergistic capacity to reduce circulating bile acid concentration, thus reflecting its potential to treat both cholestatic diseases (such as PBC and PSC) and fibrotic diseases.